Adeno-associated virus (AAV) is a single-stranded human DNA parvovirus whose genome has a size about of 4.6 kb. The AAV genome contains two major genes: the rep gene, which codes for the rep proteins (Rep 76, Rep 68, Rep 52 and Rep 40) and the cap gene, which codes for AAV structural proteins (VP-1, VP-2 and VP-3). The rep proteins are involved in AAV replication, rescue, transcription and integration, while the cap proteins form the AAV viral particle. AAV derives its name from its dependence on an adenovirus or other helper virus (e.g., herpesvirus) to supply essential gene products that allow AAV to undergo a productive infection, i.e., reproduce itself in a host cell. In the absence of helper virus, AAV integrates as a provirus into a host cell's chromosome, until it is rescued by superinfection of the host cell with a helper virus, usually adenovirus (Muzyczka, Curr. Top. Micro. Immunol. 158:97-127, 1992).
Interest in AAV as a gene transfer vector (recombinant AAV, rAAV) results from several unique features of its biology. At both ends of the AAV genome is a nucleotide sequence known as an inverted terminal repeat (ITR), which contains cis-acting nucleotide sequences required for virus replication, rescue, packaging and integration. The integration function of the ITR, mediated by AAV rep proteins in trans, permits the AAV genome to integrate into a cellular chromosome after infection, in the absence of helper virus. This unique property of the virus has relevance to the use of rAAV in gene transfer, since it allows for the integration of a recombinant AAV containing a foreign nucleic acid (transgene) into the cellular genome. Therefore, stable genetic transformation, a major goal of gene transfer, may be achieved by use of rAAV vectors. Furthermore, the site of integration for AAV is well-established and has been localized to chromosome 19 of humans (Kotin et al., Proc. Natl. Acad. Sci. USA 87:2211-2215, 1990). This predictability of integration site reduces the danger of random insertional events into the cellular genome that may activate or inactivate host genes or interrupt coding sequences, consequences that can limit the use of vectors whose integration is random, e.g., retroviruses. However, because the rep proteins mediate the integration of AAV, removal of the rep gene in the design of rAAV vectors may result in the altered cellular integration patterns that have been observed with rAAV vectors (Ponnazhagan et al., Hum. Gene Ther. 8:275-284, 1997).
There are additional advantages to the use of AAV for gene transfer. The host range of AAV is broad. Moreover, unlike retroviruses, AAV can infect both quiescent and dividing cells. In addition, AAV has not been associated with human disease, obviating many of the concerns that have been raised with retrovirus-derived gene transfer vectors.
Progress in the development of AAV as a gene transfer vector, however, has been limited by an inability to produce high titer rAAV stocks. Standard approaches to the generation of rAAV vectors have required the coordination of a series of intracellular events: transfection of a host cell with an rAAV vector genome containing a transgene of interest flanked by the AAV ITR sequences, transfection of the host cell by a plasmid encoding the AAV rep gene whose protein products are required in trans, and infection of the transfected cell with a helper virus to supply the non-AAV helper functions required in trans (Muzyczka, N., Curr. Top. Micro. Immunol. 158: 97-129, 1992). The adenoviras (or other helper virus) proteins activate transcription of the AAV rep gene, and the rep protein products thereof then activate transcription of the AAV cap gene. The cap proteins then utilize the ITR sequences to package the rAAV genome into a virus particle.
The efficiency of packaging is determined, in part, by the availability of adequate amounts of the structural proteins (VP-1, VP-2, VP-3), as well as by the accessibility of any cis-acting packaging sequences required in the rAAV vector genome.
One of the potential limitations to high level rAAV production derives from limiting quantities of the AAV helper proteins (those encoded by the AAV rep and cap genes) required in trans for replication and packaging of the rAAV genome. Various approaches to increasing the levels of these proteins have included placing the AAV rep gene under the control of the HIV LTR promoter to increase the levels of rep proteins produced (Flotte, F. R. et al., Gene Therapy 2:29-37, 1995); the use of other heterologous promoters to increase production of the AAV helper proteins, specifically the cap proteins (Vincent et al., J. Virol. 71:1897-1905, 1997); and the development of cell lines containing the AAV rep gene that specifically produce the rep proteins (Yang, Q. et al., J. Virol. 68: 4847-4856, 1994).
Other approaches to improving the production of rAAV vectors include the use of helper virus induction of the AAV helper proteins (Clark et al., Gene Therapy 3:1124-1132, 1996) and the generation of a cell line containing integrated copies of the rAAV vector and AAV helper genes such that infection by the helper virus initiates rAAV production (Clark et al., Human Gene Therapy 6:1329-1341, 1995).
rAAV vectors have also been produced using either replication-defective helper adenoviruses which contain within their genome nucleotide sequences encoding a rAAV vector genome (U.S. Pat. No. 5,856,152 issued Jan. 5, 1999) or helper adenoviruses whose genomes contain nucleotide sequences encoding AAV helper proteins (PCT International Publication WO95/06743, published Mar. 9, 1995). Production strategies which combine high level expression of the AAV helper genes and the optimal choice of cis-acting nucleotide sequences inserted into a rAAV vector genome have been described (PCT International Application No. WO97/09441 published Mar. 13, 1997).
A further limitation to the production of high titer, purified rAAV stocks is that the requirement for the presence of a non-AAV helper virus can can lead to contamination of a vector stock preparation with helper virus. The helper virus can itself replicate during rAAV production, thereby requiring further technical manipulations of a cell lysate in order to produce a purified stock of rAAV vectors. In addition to the inefficiency in such purification techniques, the contamination by helper viruses is ultimately undesirable for therapeutic applications of rAAV as gene transfer vectors. Current approaches to reducing contamination of rAAV vector stocks by helper viruses, include, inter alia, the use of temperature-sensitive helper adenoviruses (Ensinger et al., J. Virol. 10:328-339, 1972), which are inactivated at the non-permissive temperature, used for producing rAAV stocks. Alternatively, the non-AAV helper genes can be subcloned into DNA plasmids which are transfected into a cell during rAAV vector production (Salvetti et al., Hum. Gene Ther. 9:695-706, 1998; Grimm et al., Hum. Gene Ther. 9:2745-2760, 1998).
The development of further rAAV production methods, which eliminate or reduce the replication of helper virus in the production of vector stocks and allow the generation of purified vector preparations, increases the feasibility of using rAAV for gene transfer. The present invention provides such methods.